Hybrid plants have become increasingly important in various commercial food crops around the world. Hybrid plants have the advantages of higher yield, better quality and stress resistance than their parents, because of heterosis or hybrid vigor. Crop uniformity is another advantage of hybrid plants when the parents are homozygous; this leads to improved crop management. Hybrid seed is therefore commercially important and sells at a premium price. In crops such as maize, sunflower, sorghum, sugar beet, cotton, and many vegetables, hybrids account for a large share of the seed market. Not only the USA and Europe, but also many developing countries rely on their food production to a large extent on hybrids. Sale of hybrids in various crops account for nearly 40 percent of the global commercial seed business of about US$ 15 billion. This share is likely to increase as the importance of hybrid vigor is yet to be realized fully, especially in developing countries.
The production of hybrid varieties of maize (from the thirties in the US), cotton (since 1970 in India) and of rice (since 1976 in China) represents the most significant and successful breeding efforts of the twentieth century. A 6-fold increase was observed between 1930 and 1990 for US corn yield after the introduction of hybrid breeding, compared to uniform performances for selected open pollinated populations during the previous 60 years (Stuber, 1994).
The concept of hybrid vigor ((Zirkle, 1952)) emerged since the early observations in the eighteenth century by J. G. Koelreuter of interspecific crosses in Nicotiana, Dianthus, Verbascum, Mirabilis, Datura, confirmed by Darwin (Darwin, 1876) in vegetables, and W. J. Beal in maize (Beal, 1880). Subsequently, this effect was exploited in plant breeding (Shull, 1952) when the tools to produce the necessary amount of seeds became available in hermaphrodite species: the first male sterility system was developed in onion in 1943 (Jones, 1943) and others were developed in a wide range of species such as sugar beet, maize, sorghum, sunflower, rice, rapeseed, carrot (Frankel, 1977).
The key to the successful commercial production of hybrid seeds is sufficient control of the pollination process that is male sterility. Male sterility is defined as the failure of plants to produce functional anthers, pollen, or male gametes. First documentation of male sterility came in 1763 when Kolreuter observed anther abortion within species and specific hybrids. Maize has distinctly separate male and female flowers which makes the plant well suited to manual or mechanical emasculation. The tassels are removed from the seed plants before they are able to shed pollen. Even though detasseling is currently used in hybrid seed production for plants such as maize, the process is labor-intensive and costly, both in terms of the actual detasseling cost and yield loss as a result of detasseling the female parent.
Most major crop plants of interest have both functional male and female organs within the same flower, therefore, emasculation is not a simple procedure. While it is possible to remove by hand the pollen forming organs before pollen is shed, this form of hybrid production is extremely labor intensive and expensive. Seed is produced in this manner only if the value and amount of seed recovered warrants the effort. A1
Another general means of producing hybrid seed is to use chemicals that kill or block viable pollen formation. These chemicals, termed gametocides, are used to impart a transitory male-sterility. Commercial production of hybrid seed by use of gametocides is limited by the expense and availability of the chemicals and the reliability and length of action of the applications. A serious limitation of gametocides is that they have phytotoxic effects, the severity of which is dependent on genotype. Other limitations include that these chemicals may not efficiently reach the mail reproductive parts or may not be effective for crops with an extended flowering period because new flowers produced may not be affected. Consequently, repeated application of chemicals is required.
Many current commercial hybrid seed production systems for field crops rely on a genetic means of pollination control. Plants that are used as females either fail to make pollen, fail to shed pollen, or produce pollen that is biochemically unable to affect self-fertilization. Of more widespread interest for commercial seed production are systems of pollen-control-based genetic mechanisms causing male sterility. There are three main types of male sterility observed in nature. All three types of male sterility are used in commercial breeding programs to ensure cross-pollination to produce hybrid seeds in different crops.
One type of male sterility is nuclear encoded called as genetic male sterility. It is ordinarily governed by a single recessive gene, ins but dominant genes governing male sterility are also known e.g. in sunflower. Thus nuclear male sterility can be either dominant or recessive. Many different nuclear male sterile (ms) genes have been isolated in maize. In rice 25 ms are known. In a plant homozygous recessive for such a gene, the pollen fails to develop to maturity. For breeding purposes, a recessive male-sterile parent plant is maintained by crossing it with a heterozygous male-fertile plant that also includes the recessive male-sterility allele, so that the offspring are 50% recessive male-sterile plants. The other 50% are male-fertile plants that have to be rogued out in outcrossing programs which can only be done efficiently if the recessive male-sterility allele is segregated together with a selectable or screenable marker. In U.S. Pat. No. 4,727,219, a procedure is described for the use of recessive male sterility for the production of, hybrid maize. Dominant nuclear male sterile plants, as compared to recessive male sterile plants, can be maintained through crossing with a male-fertile plant, to produce offspring that are 50% dominant male-sterile plants. The usefulness of dominant nuclear male-sterile plant is, however, limited because its dominant male-sterility allele is in most cases not tightly linked (i.e., within the same genetic locus) to a selectable or screenable marker. Dominant sterility can only be used for hybrid seed formation if propagation of the female line is possible (for example, via in vitro clonal propagation). Dominant nuclear male-sterile lines were developed with a blue seed marker in durum and common wheat (Tian and Liu, 2001). This genetic male sterility is of wide occurrence in plants but commercial utility of this sterility system is limited by the expense of clonal propagation and roguing the female rows of self-fertile plants.
Genetic male sterility may be subdivided into two broad groups: (1) environment insensitive i.e. ins gene expression is much less affected by environment and (2) environment sensitive i.e. ins gene expression occurs within specific range of temperature and/or photoperiod regimes; this type of sterility is known in rice, tomato, wheat etc. The environment sensitive male sterility is further divided into two groups (1) temperature sensitive genetic male sterility e.g. rice TGMS line Pei-Ai645 and (2) photoperiod sensitive genetic male sterility e.g. rice 5047S. In addition approaches in genetic engineering have been used to produce transgenic male sterility, for which a novel approach is discussed in this document.
The second type of male sterility is conditioned by hereditary particles in the cytoplasm. Cytoplasmic male sterility is caused by the extranuclear genome (mitochondria or chloroplast) and shows maternal inheritance. Manifestation of male sterility in these may be either entirely controlled by cytoplasmic factors or by the interaction between cytoplasmic and nuclear factors. They show non-Mendelian inheritance. This is not a very common type of male sterile system in the plant kingdom. Cytoplasmic male sterility (CMS) of the seed line can be achieved through crossing with naturally occurring CMS germplasm as female parent. Here the sterility is transmitted only through the female and all progeny will be sterile. This is not a problem for crops such as onions or carrots where the commodity harvested from the F1 generation is produced during vegetative growth. But in other cases where clonal propagation is not possible CMS lines must be maintained by repeated crossing to a sister line (known as the maintainer line) that is genetically identical except that it possesses normal cytoplasm and is therefore male fertile. This approach of induction of male sterility in the seed line on the basis of sterilizing cytoplasm was employed in rice, sorghum, sunflower and millet. But the offspring of plants of this type are only of commercial value if the economic product of the offspring is not for use as seed but rather for plants such as ornamentals and sugarbeet.
When nuclear genes for fertility restoration (Rf) are available for CMS system in any crop, it is called as cytoplasmic genetic male sterility (CGMS). The restorers of fertility (Rf) genes are distinct from genetic male sterility genes. This third type male sterility system is the result of a combination of both nuclear encoded male sterility and cytoplasmatically encoded male sterility. Here sterility is manifested by the influence of both nuclear and cytoplasmic genes. The cases of cytoplasmic male sterility would be included in the cytoplasmic-genic system as and when restorer genes for them would be discovered. It is likely that a restorer gene would be found for all the cases of cytoplasmic male sterility if thorough search were made. There are commonly two types of cytoplasms, N (normal) and S (sterile). The Rf genes do not have any expression of their own unless the sterile cytoplasm is present. Rf genes are required to restore fertility in S cytoplasm which causes sterility. Thus a combination of N cytoplasm with rfrf and S cytoplasm with Rf-produces fertiles; while S cytoplasm with Of produces only male steriles. N cytoplasm with Rfrf is best for stable fertility. U.S. Pat. No. 6,320,098 described a method of producing cytoplasmic-genetic male sterile soybean and method for producing hybrid soybean. U.S. Pat. No. 5,773,680 utilized cytoplasmic-genetic male sterility system in the production of hybrid wild rice.
Generally, the use of CMS for commercial seed production involves maintenance of three breeding lines: a male-sterile line (female parent), a maintainer line which is isogenic to the male-sterile line but contains fully functional mitochondria and a restorer line which has nuclear genes (Rf genes) for fertility restoration.
Discovery of dominant negative genes which would alter plant development would be particularly useful in developing genetic methods to induce male sterility because other available methods, including cytoplasmic male sterility and nuclear male sterility have shortcomings. A dominant negative gene is one that, when expressed, effects a dominant phenotype in the plant. Herskowitz (1987), used the term “dominant negative” to denote a gene that encodes a mutant polypeptide which, when over-expressed, disrupts the activity of the wild-type gene. A wild type gene is one from which the mutant derived. In the present description the dominant negative gene is applied to a gene coding for a product that disrupts an endogenous genetic process of a host cell which receives the gene, and that is effective in a single copy or may produce an effect due to overexpression of the gene either by increased production of the gene product. Exemplary of the class of dominant negative genes are cytotoxic genes, methylase genes, and growth-inhibiting genes. Dominant negative genes include diphtheria toxin A-chain gene (Czako and An, 1991), cell cycle division mutants such as CDC in maize (Colasanti, et al., 1991) the WT gene (Farmer, et al., 1994) and P68 (Chen, et al., 1991). Biotechnology has enabled the development of several new pollination control systems that could be useful for hybrid seed production. Since the first transgenic male sterility system was described (Mariani, 1990), many strategies to produce male-sterile plants have been reported. There has been significant interest in using an ablation system for controlling reproductive development in plants. Reproductive control has been achieved in several plant species by genetic ablation, which entails linking a reproductive-preferred promoter with a dominant negative gene to ablate reproductive cells. Prior art regarding the proposed invention are as follows:                Patents EP344029, EP1135982 and WO89/10396 described a system for producing a male sterile plant by transforming a plant with a DNA encoding barnase under the control of a tapetum-specific promoter. Barnase is an RNase originating in Bacillus amyloliquefaciens. This enzyme has 110 amino acid residues and hydrolyzes RNA. When expressed in cells, this enzyme degrades RNA in cells and thus inhibits the functions of the cells and finally causes cell death in many cases. By using this characteristic, it is therefore expected that the function of the specific site can be selectively controlled by expressing the barnase gene in a specific site of a plant. Transformation of tobacco and oilseed rape plants with such a promoter-gene construct prevented the plants from producing fertile pollen (Mariani et al., 1990). Similarly collapse of tapetum was also observed when A9 and A6 promoters were used to drive expression of the barnase gene in transgenic plants (Hird et. al., 1993; Paul et. al., 1992).        When the barnase gene was employed as a male sterility gene, however, it was frequently observed that resulting male sterile transgenic plants exhibit unfavorable characteristics. PCT International Publication WO96/26283 refers to this problem in rice. It is also reported that similar phenomena are observed not only in rice but in lettuce (Reymaerts et. al., 1993). Patent Application 20020166140 reported mutated barnase gene at least in part and then the thus obtained mutant barnase gene, having a weakened effect was anther-specifically expressed in a plant so as to make the plant substantially male sterile without any substantially disadvantageous effect on the tissues other than anthers. In this patent production of male sterile plants, free from any unfavorable characteristic at a high efficiency was claimed.        U.S. Pat. No. 5,763,243, U.S. Pat. No. 6,072,102, U.S. Pat. No. 5,792,853, U.S. Pat. No. 5,837,851 and U.S. Pat. No. 5,795,753 have used a DNA adenine methylase (DAM) gene, isolated from E. coli as a dominant negative gene. Changes in the DNA methylation pattern of specific genes or promoters have accounted for changes in gene expression. Methylation of DNA is a factor in regulation of genes during development of both plants and animals. Methylation patterns are established by methods such as the use of methyl-sensitive CpG-containing promoters (genes). In general, actively transcribed sequences are under methylated. In animals, sites of methylation are modified at CpG sites (residues). Genetic control of methylation of adenine (A) and cytosine (C) (nucleotides present in DNA) is affected by genes in bacterial and mammalian species. In plants, however, methyl moieties exist in the sequence CXG, where X can be A, C or T, where C is the methylated residue. Inactivation due to methylation of A is not known in plants, particularly within GATC sites known to be methylated in other systems. E. coli DNA adenine methylase (DAM) for which GATC is a target inactivates a genetic region critical for pollen formation or function thereby causing a male sterile plant to form.        Patent E P0942965, U.S. Pat. No. 6,177,616 and U.S. Pat. No. 6,384,304 used DNA molecules which code for deacetylases or proteins having the biological activity of a deacetylase. These molecules can be used to produce plants having parts which can be deliberately destroyed i.e. plants which have male sterility, by the specific expression of a deacetylase gene (Kriete et. al. 1996, Bartsch 2001). The deacetylase genes from Streptomyces viridochromogenes [N-acetyl-L-phosphinothricylalanylalanine (N-acetyl-PTT) deacetylase, dea] and argE from Escherichia coli (N-acetyl-L-ornithine deacetylase) encode proteins having specificity for N-acetyl-L-PPT. For both genes, it was possible in the case of tapetum-specific expression in plants to show the occurrence of male-sterile flowers after treatment of individual buds with N-acetyl-L-PPT. For successful use of this system, in particular in the treatment of whole plants with N-acetyl-PPT under practically relevant conditions, it is advantageous to be able to employ deacetylases having high substrate affinity. Therefore further deacetylases having high affinity for N-acetyl-PPT were sought. In U.S. Pat. No. 6,177,616 and U.S. Pat. No. 6,384,304 N-acetyl-PPT deactylase gene from Stenotrophomonas sp. was used for the production of male sterile plants.        Patent E P0455690, reported a method of inhibiting respiration of a plant cell by use of a gene, which is expressible in anthers of plants, to inhibit mitochondrial function leading to cell death and failure to produce viable pollen, thus imparting male sterility. The disrupter gene was selected from the mammalian uncoupling protein (UCP) cloned from mammalian (usually rat) brown adipose tissue. The proposed disrupter protein, UCP, is instrumental in the thermogenesis of mammalian brown adipose tissue and exists as a dimer in the mitochondrial inner membrane forming a proton channel and thus uncoupling oxidative phosphorylation by dissipation of the proton electrochemical potential differences across the membrane.        U.S. Pat. No. 5,254,801, reported a phosphonate monoesterase gene (pehA), found suitable for purpose such as inducing male sterility for hybrid seed production in plants. A bacterial phosphonate monoester hydrolase was evaluated in plants as a conditional lethal gene useful for cell ablation and negative selection. A phosphonate monoesterase gene (pehA) encoding an enzyme that hydrolyzes phosphonate esters including glyceryl glyphosate to glyphosate and glycerol was cloned from the glyphosate metabolizing bacterium, Burkholderia caryophilli PG2982. As an example of tissue-specific cell ablation, floral sterility without vegetative toxicity was demonstrated by expressing the pehA gene using a tapetum specific promoter and treating the mature plants with glyceryl glyphosate. (Dotson et. al. 1996).        WO 99/04023 proposed a method of controlling fertility of plants by the use of DNA molecule that encodes avidin, a glycoprotein. High level expression of avidin gene in anthers can induce male sterility. Avidin, a glycoprotein has a very strong affinity for biotin (vitamin H) with a KD (dissociation constant) of approximately 10−15 M−1[1], the highest known affinity between any protein and its ligand. This binding is essentially irreversible. Fertility can be restored by spraying the plant with a solution of biotin.        U.S. Pat. No. 5,955,653 discovered a tapetum-specific callase (beta.-1,3-glucanase) gene, designated A6, from Brassica napus and other members of the family Brassicaceae including A. thaliana. The A6 gene encodes a 53 kDa callase enzyme of Brassica napus and equivalent proteins in other Brassicaceae family members. Coding sequence from the gene can be driven by an appropriate promoter to induce male sterility in plants. Microspore release is the process by which the immature microspores are liberated from a protective coat of .beta.(1,3) poly-glucan (callose) laid down by the microsporogenous cells before meiosis (Rowley, (1959); Heslop-Harrison (1968)). The anther-expressed glucanase responsible for the dissolution of this callose coat is known as callase. Callase is synthesised by the cells of the tapetum and secreted into the locule. The appearance of the enzyme activity is developmentally regulated to coincide precisely with a specific stage of microspore development. The basis of the use of a glucanase as a sterility DNA lies in the fact that mis-timing of the appearance of callase activity is associated with certain types of male-sterility (Warmke and Overman, 1972). One important attraction of glucanase as a potential sterility DNA is that it already occurs in a natural system. But the timing of the appearance of callase activity is critical.        U.S. Pat. No. 7,230,168 described transformation of a plant cell with a nucleic acid construct encoding cytokinin oxidase where expression of the cytokinin oxidase inhibits pollen formation or male organ development in the transgenic plant. Fertility restoration in the plant may be achieved after restoration of normal cytokinin levels by application of cytokinins or cytokinin oxidase inhibitor such as a cytokinin oxidase 1 inhibitor. Hear ability of the particular cytokinin oxidase to oxidatively remove cytokinin side chains to give adenine and the corresponding isopentenyl aldehyde was utilized to create male sterility.        In animal systems, studies of apoptosis have revealed pathways where proteins of the Bcl-2 family play key roles. The Bcl-2 family includes pro-apoptotic (e.g. Bax, Bak and Bid) and anti-apoptotic (e.g. Bcl-2, Bcl-xl and Ced-9) members that appear to control the initiation of apoptosis through mitochondria (Gross et al. 1999). A Bax gene has been shown to induce PCD in plant cells (Lacomme and Cruz 1999, Kawai-Yamada et al. 2001). A mouse Bax gene was connected to the tapetum-specific promoter, expression of the Bax gene caused cell death resulting pollen abortion (Tsuchiya et al. 1994, Ariizumi et al. 2002). A suppressor of Bax-induced cell death has been identified in plants. Expression of AtBI-1, a homolog of mammalian Bax inhibitor, in the tapetum at the tetrad stage inhibits tapetum degeneration and subsequently results in pollen abortion, while activation of AtBI-1 at the later stage does not (Patent JP2006345742-A, Kawanabe et. al. 2006).        Diphtheria toxin A chain (DTA) gene was expressed in tapetum which resulted in dominant male sterility due to the specific cell ablation (Koltunow et. al., 1990). Similarly, when the S-locus glycoprotein gene promoter of Brassica was fused to the DTA gene and transferred into tobacco (Thorness et al., 1991) and A. thaliana (Thorness et al 1993) it resulted in self-sterile plants due to expression of gene in both pistil and anthers. APETALA3 (AP3) promoter-DTA fusion resulted in the complete ablation of petals and stamen in transgenic tobacco (Day et. al., 1995). Temperature sensitive diphtheria toxin A chain (DTA) gene was also used to confer conditional male sterility in Arabidopsis thaliana (Guerineau F et. al., 2003).        O'Kefee et al (1994) described R7402/P450sU1 system in which P450SU1 (Streptomyces griseolus gene encoding herbicide-metabolizing cytochrome) expression and R7402 treatment can be used as a negative selection system in plants. In tobacco expressing P450SU1 from a tapetum-specific promoter, treatment of immature flower buds with R7402 caused dramatically lowered pollen viability. Such treatment could be the basis for a chemical hybridizing agent. This may provide a strategy for development of a chemical male sterilant for hybrid seed production.        A ribosome inactivating protein (RIP) from D. sinensis was used as a cytotoxic gene to induce male sterility in tobacco plants (Cho H J et. al. 2001). Ribosome inactivating protein inactivates eukaryotic ribosomes and inhibits general protein synthesis. Actually it inhibits its own protein synthesis (Boness et. al. 1994). Due to its suicidal action it was proposed to use in genetic cell ablation and genetic improvement by Cho H J.        Hofig et. al. (2006) expressed a stilbene synthase gene (STS) in anthers of transgenic Nicotiana tabacum plants, resulting in complete male sterility in 70% of transformed plants. The grapevine stilbene synthase (STS) has been shown to compete with the enzyme chalcone synthase (CHS) for the substrates malonyl-CoAand coumaroyl-CoA. STS-induced sterility in tobacco is believed to result from a reduced or abolished flavonol biosynthesis. This has been confirmed by experiments where STS-sterile tobacco plants were regularly sprayed with flavonols and where fertility was partially restored. STS, when expressed in non-tapetal cells, is not expected to haye a toxic impact since there is no competing CHS present.        
Autophagy is a ubiquitious process in eukaryotic cells, in which portions of the cytoplasm are sequestered in double-memberane vescicles for delivery to a degradative organelle, vacuole or lysosome (Reggiori et. al. 2002). Autophagy is known to be active at basal levels under normal physiological conditions; it can be stimulated by a plethora of stresses including cellular damage, nutrient starvation and pathogen infection (Levine and Klionsky, 2004). It is well established that autophagy promotes cell survival during nutrient starvation by degrading and recycling nutrients (Seay M. et. al. 2006). AuTophaGy-related (ATG) genes are essential for autophagosome formation. In the last decade, with the identification of approximately 30 ATG genes in Saccharomyces cerevisiae and other fungi (Klionsky et. al. 2003), the molecular mechanisms of autophagy have gradually been elucidated ((Klionsky et. al. 2005). Autophagy is conserved across all eukaryotes and homologs of many yeast ATG genes have recently been identified in various eukaryotic systems, and the molecular mechanisms of autophagy are also conserved (Yang Cao et. al. 2007). Autophagosome formation is a complex process and each Atg protein has been shown to function at specific stage during autophagosome formation in the yeast (Tsukada et. al. 1993). A number of Atg proteins accumulate to a perivacuolar structure termed the pre-autophagosomal structure (PAS) (Kim et. al. 2002). Among the ATG genes, ATG6 is relatively unique in its not being autophagy-specific (Yang Cao et. al. 2007). For example, the S. cerevisiae ATG6/VPS30 gene product is the only protein required for both autophagy and sorting of the vacuole resident hydrolase carboxypeptidase Y through the Vps pathway (Kametaka et. al. 1998). Yeast Atg6/Vps30 is a subunit of two distinct class III phosphotidylinositol (PtdIns) 3-kinase complexes pathways (Kihara et. al. 2001). Complex I functions in autophagy, whereas complex II is involved in Vps, which explains why Atg6/Vps30 participates in both, otherwise separate, pathways.
BECLIN 1, the mammalian homologue of yeast ATG6 was the first identified mammalian gene with a role in mediating autophagy (Liang et. al., 1999). BECLIN 1 was originally discovered during the course of a yeast two-hybrid screen of a mouse brain cDNA library using human Bc1-2 as the bait (Liang et. al. 1998). Overexpression of Human BECLIN 1 prompts autophagic cell death in human MCF7 breast carcinoma cells (Liang et. al., 1999). Recently BECLIN 1 was found to participate in apoptosis signaling through caspase-9 thus BECLIN 1 may be the critical ‘molecular switch’ and play an important role to fine tune autophagy and apoptosis (Wang et. al. 2007). BECLIN 1 is conserved in higher eukaryotes. Human Beclin 1 protein shares 36% identity and 52% similarity with Nicotiana Beclin 1 (Liang et. al., 1999).
If a plant is to survive an infection, hypersensitive response (HR) cell death (PCD) must be carefully controlled so that it does not spread throughout the plant and kill it. The plant ortholog of BECLIN 1 was first studied in Nicotiana benthamiana plants (Liu Y. et. al. 2005) and it was found essential for restriction of HR PCD during disease resistance (Seay M. et. al. 2006, Liu Y. et. al. 2005, Patel S. et. al. 2006). Plants deficient in the plant BECLIN 1 exhibit unrestricted HR PCD in response to pathogen infection (Liu Y. et. al. 2005). Autophagosomes were rarely observed in the cells of plant BECLIN 1 silenced plants after infection with TMV (Liu Y. et. al. 2005). Autophagosomes are induced at the site of TMV infection during HR PCD and plant BECLIN 1/ATG6 is required for induction of autophagy in both pathogen infected cells and uninfected adjacent cells to restrict HR PCD at infected site (Liu Y. et. al. 2005). Thus there is a prodeath signal(s) moving out of the pathogen-infected area into adjacent tissues and distal sites that is negatively regulated by autophagy. These findings provide the genetic evidence that ATG genes can function in vivo as a negative regulator of HR PCD. These results contrast with findings from mammalian studies in which ATG genes are required to promote PCD in cells lacking intact apoptotic machinery (Liu Y. et. al. 2005).
Recently, it was reported that AtBECLIN 1/ATG6 in plants has distinct function in addition to autophagy: vesicle trafficking and pollen germination (Fujiki Y. et. al. 2007, Qin G. et. al. 2007). They reported that deletions of AtBECLIN 1/ATG6 specifically influenced male gametophytes but not the female reproductive structures. Pollens lacking AtBECLIN 1/ATG6 failed to germinate. During pollen germination and pollen tube growth, cellular trafficking is critical for cell wall deposition and cell shape remodeling (Parton R M et. al. 2003, Parton R M et. al. 2001, and Helper P K et. al. 2001). It is possible that ATG6 deletions alter the cellular trafficking system which results failure of pollen germination (Qin G. et. al. 2007). AtBECLIN 1/ATG6 deficient plants displayed retarted growth, dwarfism and early senescence this suggests that AtBECLIN 1/ATG6 is required for normal plant development (Qin G. et. al. 2007).
Tapetum is the innermost sporophytic layer of anther wall and surrounds the microspores. The tapetum is known to provide nutrition to developing microspores especially exine of pollen grains, the main structural components of the pollen wall. The tapetum degenerates during the later stages of pollen development. It has been speculated that tapetum degeneration is a programmed cell death (PCD) event (Wu and Cheun 2000). [Nuclei of tapetum cells and the tissues of anther wall were found TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling) positive by Wang et. al. (1999).] The proper timing of cell death in the tapetum is essential for normal microsporogenesis. Kawanabe et. al. (2006) had shown that expression of mouse Bax gene in tapetum at early stage of pollen development can cause early degeneration of tapetum resulting into pollen abortion.
In the present invention, AtBECLIN 1/ATG6 gene is being expressed in tapetum in stage 2 and 3 of pollen development (which has not been previously reported). This causes disruption of normal cell death programme of tapetum and there is a delay in the induction of tapetal programmed cell death (PCD). Hence, pollen formed are abnormal having an intact tapetum, resulting in male sterility.